This project characterizes the structure and function of bacterial protein toxins to determine how toxins contribute to bacterial pathogenesis. Sensitive eukaryotic cells are studied to identify subcellular systems with which the toxins interact. (A) In order to intoxicate cells the anthrax toxin protective antigen (PA) must be activated by cleavage after the amino acid sequence RKKR. Mutant animal cell lines were derived that are deficient in proteases that normally activate PA. Several of these mutant cell lines appear to lack the protease furin, which is known to recognize sequences having R at the -1 and -4 positions, as in the sequence RAAR. Thus, the mutant cells are resistant to a PA variant having the cleavage sequence RAAR and to Pseudomonas exotoxin, which requires proteolytic activation at the sequence RQPR. Other mutant cell lines were obtained that are resistant to PA variants having the sequence SSRR. Evidence suggests these cell lines lack a protease that recognizes the RR sequence. (D) Improved methods were developed for production and purification of fusion proteins containing amino acids 1-254 of the anthrax toxin LF protein and cytotoxic catalytic domains from other toxins. These proteins are efficiently translocated by the anthrax toxin PA protein to the cytosol of animal cells where they cause cell death. The fusion proteins are being developed as one component of therapeutic, cell-type specific cytotoxic agents. (C) Strong evidence was obtained that the anthrax toxin LF protein is a metalloprotease. A sequence HEXXH, shared by all metalloproteases, was found in the LF sequence. Site-directed mutagenesis to change either of the His residues to Ala completely destroyed toxicity. Furthermore, a number of protease and peptidase inhibitors protected cells against the toxin. The results have wider interest because tetanus toxin and the botulinum neurotoxins were recently shown to be metalloproteases. (D) A Bacillus anthracis gene was identified that is needed to stimulate anthrax toxin synthesis. Sequencing showed the gene encodes a protein of 476 amino acids. No similar proteins were found in existing databases so this gene may be representative of a new class of regulator.